The selection of efficient and economic downstream sequences for purification of polypeptides produced by recombinant DNA technology is a crucial step in the development of every new biopharmaceutical intended for therapeutic use. In the recent past the need for large scale purification processes for monoclonal antibodies (mabs), due to their exceptionally high therapeutic dosages in medical use, has been further intensified with the use of improved cell culture methods resulting in higher cell densities and higher expression rates. The increasing concentrations in the culture fluids of product and contaminants set higher demands on the capture chromatography, on its preceding sample clarification steps and on the subsequent polishing chromatographies. The entire downstream process has to: (i) manage an increased mass of product, (ii) efficiently remove increased process- and product-related impurities to below defined acceptance criteria, and (iii) maintain economic yields and sufficient quality of the mab. Usually, the downstream process accounts for a major part of the total manufacturing costs of therapeutic antibodies.
The mabs in crude fractions are typically associated with impurities such as host cell proteins (HCP), host cell DNA, viruses, aggregates, other undesired product variants, and various leachates from process materials. The presence of these impurities is a potential health risk for patients, and hence their absence from the final product is a regulatory requirement. Only very low residual amounts will be tolerated.
The classical procedure for purifying cell-culture derived polypeptides follows the sequence of capture-intermediate-polishing chromatographies, accompanied by filtration, concentration or dialysis steps at various positions of the downstream sequence. In recent years platform approaches have been successfully established in the field of mab purification. Since mabs are a well-defined class of glycoproteins possessing common physicochemical properties, the use of a generic platform process is reasonable (Kelly B 2009). Such a universal process, with more or less product-specific adaptions, can be applied to many mabs, especially for those immunoglobulins of the same class or subclass, e.g. IgG1.
One of the most frequent capture steps used for mab purification is affinity chromatography with Protein A. This capture offers exceptional selectivity for Fc-bearing molecules, thereby removing more than 99.5% of contaminants in a single step. However, besides its advantages, two disadvantages should also be mentioned. One drawback is the undesired leaching of Protein A or fragments of Protein A which are known to be toxic (Gagnon P 1996). The other disadvantage is the high cost of this type of resin, particularly at the industrial scale necessary to purify therapeutic antibodies. A Protein A resin is approximately 30 times more expensive than an ion exchange resin. It was calculated that for the downstream processing of a 10 m3 cell culture the cost for the Protein A affinity chromatography is about 4-5 million USD (Farid SS 2009).
Many solutions have been published to overcome the problem of leached Protein A (Gagnon P 1996; Fahrner R L 2001). Several approaches related to post-Protein A chromatographic steps which remove leached Protein A, such as anion exchange chromatography used in binding mode (EP0345549) or flow-through mode (WO2004076485), cation exchange chromatography (WO2009058812), hydrophobic interaction chromatography (WO9522389), or combinations of chromatographies, for instances ion exchange chromatography followed by hydrophobic interaction chromatography (WO2010141039), anion exchange chromatography followed by cation exchange chromatography (WO2011090720), or cation exchange chromatography and Mixed Mode chromatography in any order (WO2011150110). Since the required overall degree of purity for a therapeutic antibody is extremely high, a typical platform purification scheme consists of at least two post-Protein A chromatographies which are usually selected from cation exchange chromatography, anion exchange chromatography in flow-through, and hydrophobic interaction chromatography (Fahrner R L 2001, Kelly B 2009, WO9522389, WO2009138484, WO2010141039, WO2011017514, WO2011090720).
Other approaches reduce the leachates already during the Protein A affinity chromatography by using special wash steps removing leached Protein A prior to eluting the immunoglobulin. Many intermediate wash buffers were developed containing salts or additional components, for example hydrophobic electrolytes such as tetramethylammonium chloride (Fahrner R L 2001).
Some methods take effect closer to the source of the Protein A leaching by directly reducing the proteolytic activities originating from the sample. A major part of Protein A leaching is caused by proteolysis. Such reduced leaching was achieved by low temperatures and/or by adding protease inhibitors to the buffers (WO2005016968).
A special method for avoiding or reducing Protein A leaching comprises pre-treatment of the Protein A resin with surface active compounds, for example chaotropic substances such as Urea or Guandine-HCl (WO03041859).
It has been known for a long time that different types of Protein A resins display different degrees of leaching (Fuglistaller P 1989). Thus the selection of the Protein A material is an important factor. Besides the ligand itself, also the backbone matrix influences the leaching, the binding capacity, and the flow rates, (Fahrner R L 2001). These parameters taken together define the column size, the process time, and thus the economy of the affinity capture step. Moreover, during the previous decade chromatography suppliers have developed more robust Protein A ligands provided by genetic engineering of the natural Staphylococcus aureus Protein A sequence. These improved resins consist of a rigid matrix in combination with an improved recombinant ligand protein specially engineered to enable alkali tolerance, high binding capacity and low ligand leakage. One example is MabSelect SuRe™ from GE Healthcare Life Sciences (WO2009138484). This material can be rapidly and efficiently cleaned after the run with up to 0.5 M NaOH. However, these benefits come at a price. MabSelect SuRe and comparable modern resins are considerably more expensive than the previous Protein A resin generation. Therefore, despite these new affinity media, there is no economic benefit, rather the opposite is true. In view of the very high costs associated with Protein A-based affinity capture, it is not surprising that alternative strategies have been developed which completely avoid any use of an affinity chromatography for purification of immunoglobulins. One example is the use of high-performance tangential flow filtration in combination with non-affinity chromatographies such as anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography or Mixed Mode chromatography (WO03102132).
In many cases, capture steps are performed with crude input (load) materials, which can cause the contamination of (accumulation of impurities on) the affinity column resin. In absence of a proper regeneration step, this can prevent successful re-use of the capture resin. In case of the affinity capture with Protein A, it has to be emphasized that ligand leaching is not the major factor in limiting the life time of the Protein A resin. The contaminants in crude culture fluids, like lipids, oxidants, aggregates or particles, metal ions and other substances promote fouling of the resins. Besides direct effects on the Protein A binding moieties, also the matrix can be irreversibly contaminated. Reduced capacities and flow rates from run to run are the consequence. This problem is not limited to Protein A resins: fouling of chromatographic resins over their operational lifetimes is a general significant problem for commercial bioseparations. Hydrophobic ligands used for hydrophobic interaction chromatography and Mixed Mode chromatography, when used as capture steps for cell culture-derived immunoglobulins, are especially susceptible for trapping lipophilic contaminants from the culture fluids. Despite sophisticated protocols for post-run cleaning steps, the lifetime of a capture column is limited and depends on the number of cycles, the operating conditions for running and cleaning, and the purity of the sample.
Mixed Mode chromatography was described mainly as an option for a polishing step downstream to Protein A (Kelly, B. 2009, WO03102132). The use of Capto MMC in the binding mode for purification of mab is known. Special elution conditions were developed (WO2011049798). Likewise, it was shown that CaptoAdhere, preferably in the flow-through mode, is a suitable polishing step after a flow-through anion exchange chromatography performed after a Protein A affinity chromatography (WO2013066707). Furthermore, some different Mixed Mode resins were investigated in an overload and elute chromatography mode and CaptoAdhere was most preferred (WO2013067301).
To clarify the heavily contaminated culture fluids, mechanical separation steps have been employed which remove most of cell debris and aggregates. Centrifugation and filtration are the most common pre-treatment steps performed prior to load of the sample to the capture resin. For large volumes, centrifugation is performed by cell separators and the filtration steps are performed by depth filters and/or micro filters. The resulting culture fluid is then referred to as “clarified cell culture supernatant” (Liu H F 2010). Although the direct load of harvested culture fluid onto the Protein A resin is a frequent method of choice (Fahrner R L 2001), other platform technologies make use of the clarification steps, i.e. centrifugation, depth filtration, and/or microfiltration (Liu H F 2010, WO9522389, WO2001150110) in order to protect the capture column.
Pre-cleaning chromatographic steps performed alternatively or additionally to the centrifugation/filtration have only been reported sporadically. The use of immobilized metal (Zn2+) chelate chromatography (IMAC) in binding mode was used prior to Protein A on a very small scale (VanDamme A.-M. 1990, Bulens F. 1991). In contrast, weak anion exchange chromatography on DEAE Cellulose was used after centrifugation, filtration, and concentration and the obtained flow-through was then loaded onto Protein A (EP0550400). Finally, the advantages of depth filtration for pre-treatment of culture fluids prior to Protein A was investigated and compared to a less effective anion exchange chromatography on TMAE Fractogel in the flow-through mode (Yigsaw Y 2006).